基于PFKFB3通路抑制肿瘤有氧糖酵解的益智仁油肝靶向微乳抗肝细胞癌作用的机制研究

Primary hepatocellular carcinoma experiences high rates of morbidity and mortality in China. Traditional Chinese medicinal oils, such as Coix and Brucea seed oils, play important roles in the treatment of liver cancer patients. In the previous study conducted for this project, it was found that Alpinia oxyphylla oil (AO), when ingested as food, demonstrated significant anti-proliferation, pro-apoptosis, and other beneficial actions detrimental to liver cancer cells (both in-vivo and in-vitro)—as well as low toxicity. AO’s effectiveness appears greater than that of Coix seed oil, but the exact mechanism is unclear as further in-depth research was not pursued. Our group further designed and developed the hepat-targeted octanogalactose ester-modified sub-microemulsion of AO [Gal(oct)-AO-SEs] for the dual purposes of enhancing efficacy and reducing toxicity. Proteomics, and preliminary experimental results, suggested that the anti-cancer effect of AO could be enhanced by affecting the PFKFB3 pathway, glycolysis, and other processes, but the action mechanism has not been fully confirmed. In this study, several methods including siRNA and inhibitor interference, LC-MS/MS, RT-PCR, Western-blot, fluorescence imaging in-vivo, lentivirus transfection, and other techniques, were used. These methods elucidated the liver-targeting effect of Gal(oct)-AO-SEs, and the anti-hepatocellular carcinoma mechanism (in-vivo and in-vitro), by acting upon the PFKFB3 pathway, and inhibiting aerobic glycolysis. This study will thus provide new concepts for targeting the PFKFB3 pathway in the treatment of liver cancer.

原发性肝癌在我国发病率高、死亡率高，且发病机制复杂、治疗手段匮乏。中药油脂类成分如薏苡仁油、鸦胆子油在肝癌患者的治疗中发挥着重要作用。本课题前期研究发现药食同源的益智仁油（AO）具有强于薏苡仁油的显著体内外抗肝癌细胞增殖、促凋亡等活性和较低的毒性；课题组进一步设计并开发出具有肝靶向作用的辛酰半乳糖酯修饰的益智仁油亚微乳[Gal(oct)-AO-SEs]以达到增效减毒的目的，蛋白组学及初步实验结果均提示其可通过影响PFKFB3通路及有氧糖酵解等机制增强AO的抗癌效果，但尚未完全证实。本研究拟采用siRNA及抑制剂干扰、LC-MS/MS、RT-PCR、Western blot、活体荧光成像、慢病毒转染等多种方法阐明Gal(oct)-AO-SEs的肝靶向作用和PK特征，证实其通过作用于PFKFB3通路抑制糖酵解而发挥体内外抗肝细胞癌的作用机制。本研究将为靶向PFKFB3通路治疗肝癌提供新的思路。

本课题筛选得到多种具有抗肝癌、肠癌和乳腺癌活性的中药天然活性成分，并开展了系统的机制研究。本研究设计并制备了一种肝靶向丁基半乳糖酯修饰的AO微乳液[But(Gal)-AO-ME]，并利用CCK-8、DCFH-DA荧光显微镜、流式细胞术和无胸腺裸鼠研究了其抗肝癌活性和详细的体内分布特性，初步证实了But(Gal)-AO-ME具有较好的成药性和抑制肿瘤有氧糖酵解的新颖的作用机制。本研究制备的But(Gal)-AO-ME具有较小的粒径(26.78 ± 2.17 nm)和多分散性指数(0.067 ± 0.005)，以及较低的表面负电荷(-9.312 ± 0.018 mV)。另外，But(Gal)-AO-ME对LM3细胞的抗增殖作用最强，具有IC50值为84.90 ± 7.50 µg/mL。But(Gal)-AO-ME(50 μg/mL)的摄取为比AO微乳液(AO-ME)高出1.33倍。But(Gal)-AO-ME较AO-ME也导致了一个1.23倍的早期凋亡率的增加。同时，Cy5负载的(Gal)-AO-ME（50mg/kg）的肿瘤内荧光比Cy5负载的AO-ME高6.42倍，表明肿瘤细胞对But(Gal)-AO-ME的吸收更高。此外，But(Gal)-AO-ME（50 mg/kg）在体内显著抑制了移植肿瘤的生长，抑制率为31.46 ± 12.13%。But(Gal)-AO-ME在体内和体外均上调Bax，下调Bcl-2水平，激活caspase-3。本研究为制备有效的肝脏靶向方法提供了一种简单的方法，可应用于其他低极性中药的制备开发。另外，But(Gal)-AO-ME作为一种治疗肝癌的新制剂具有较大的开发潜力。同时，本研究为靶向肿瘤有氧糖酵解和针对PFKFB3被抑制后发挥抗肝癌的活性提供了更多的数据支持。